PROJECT 2: GENETIC IDENTIFICATION OF HOST INNATE PATHWAYS THAT CONTROL BACTERIAL PATHOGENESIS SUMMARY Phagocytes of the mammalian innate immune system, in particular macrophages and neutrophils, form the first line of defense upon bacterial infection and are armed with powerful mechanisms to limit bacterial growth and eradicate invaders. Bacterial pathogens, however, have evolved mechanisms to thwart these killing mechanisms of phagocytes, and to persist in human tissues. In addition to having direct antimicrobial activity, macrophages and neutrophils also initiate and shape powerful inflammatory responses that dramatically influence disease. Indeed, the inflammatory pathways elicited by each of the pathogens involved in this study - Mycobacterium tuberculosis (Mtb), Staphylococcus aureus (SA), and Chlamydia trachomatis (CT) - play major roles in bacterial persistence in tissues and in promoting disease. Pathogens must subvert these cells in order to grow and persist, but the host genes and cellular pathways that dictate the outcome of infection are not entirely clear. The broad idea of this proposal is to use unbiased, systematic approaches to probe the intimate interactions between pathogen and innate immune cells, and to use this information to make predictions about bacterial infectivity that will be tested in human samples in an iterative fashion to model host response during infection. We propose to systematically identify innate immune gene networks that underlie pathogenesis in clinically relevant model systems of infections with these three important bacterial pathogens. Using powerful CRISPR-based knockout strategies in both ex vivo infections and in mouse models, coupled with innovative approaches to identify functionally relevant polymorphisms in clinical samples, we seek to dramatically increase our understanding of infection biology of humans. We anticipate that these insights, coupled with the other elements of the HPMI Center, will point to novel vulnerabilities with therapeutic relevance.